Project Abstract Mutations affecting protein components of the ribosome, an organelle essential in all nucleated cells for translating mRNA, underlie a growing list of congenital diseases. It remains unclear how various germline defects in the ubiquitous ribosome cause highly dissimilar and tissue-specific pathologies. Potential clues to this conundrum lie in recent reports that physiologic variation in ribosome composition can regulate translation of specific genes, often with tissue-specific effects. This novel gene regulatory paradigm suggests a myriad of mechanisms by which ribosomes affect development and cellular physiology that await discovery. Canonically, each ribosome contains 80 ribosomal proteins (RPs), most of which are each assumed to be encoded by a single gene. However, these RP genes have hundreds of little-understood splice variants, paralogs, and pseudogenes genome-wide, some of which have open reading frames that could produce proteins partly resembling canonical RPs. I hypothesize that certain splice variants, paralogs, and pseudogenes encode alternative RP isoforms that are expressed under specific biological conditions, and form distinct ribosomes with specialized roles in mRNA translation. Such a model could uncover combinatorially numerous possibilities for ribosome diversity, and reveal functions of many poorly understood ribosomal genes. To test my hypothesis, I will first characterize the RP paralog S27L as a paradigm model system of alternative RP function. My preliminary work suggests that, during lactation, mammary luminal epithelial cells undergo a dynamic switch by downregulating canonical RP S27 and upregulating its paralog S27L. This suggests that S27L-containing ribosomes may specialize in translating genes relevant to epithelial differentiation or high- volume protein synthesis. Second, I will develop the first systematic bioinformatic and proteomic pipeline to comprehensively investigate alternative RP expression across many cell types and developmental stages. The number of potential alternative RPs is vast, as is the number of biological conditions that may require their functions. There is therefore a need for a high-throughput approach using cell type- and developmental stage- resolved transcriptomic analysis to reveal conditions under which novel alternative RPs are expressed and incorporated into ribosomes. I will subsequently use ribosome fractionation and mass spectrometry to determine whether potential alternative RP transcripts are translated and incorporated into ribosomes in primary mouse tissues. Together, these orthogonal aims set a precedent for exploring the considerable potential impact of alternative RPs on development and health. Importantly, through this work I will gain diverse expertise in cutting-edge experimental methods, computational techniques, and scientific reasoning, advancing towards my goal as a physician-scientist to elucidate genetic mechanisms underlying human disease.